EP1617249A1 - Microscope optique à balayage avec une répartition ponctuelle de la lumière et son utilisation - Google Patents

Microscope optique à balayage avec une répartition ponctuelle de la lumière et son utilisation Download PDF

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Publication number
EP1617249A1
EP1617249A1 EP04023492A EP04023492A EP1617249A1 EP 1617249 A1 EP1617249 A1 EP 1617249A1 EP 04023492 A EP04023492 A EP 04023492A EP 04023492 A EP04023492 A EP 04023492A EP 1617249 A1 EP1617249 A1 EP 1617249A1
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EP
European Patent Office
Prior art keywords
illumination
spot
sample
laser
arrangement
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP04023492A
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German (de)
English (en)
Inventor
Ralf Wolleschensky
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Carl Zeiss Microscopy GmbH
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Carl Zeiss Jena GmbH
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Publication of EP1617249A1 publication Critical patent/EP1617249A1/fr
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0064—Optical details of the image generation multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling

Definitions

  • the invention relates to a confocal laser scanning microscope with a spot illumination arrangement, which provides an illumination beam for point or point group illumination of a sample, a scanning arrangement that scans the point or point group illumination beam over the sample, a spot detector array, via the scanning arrangement images the illuminated point or point group spot of the sample by means of at least one confocal aperture on at least one detector unit, and a control unit which controls the scanning arrangement and reads the spot detector arrangement.
  • the invention further relates to a method of laser scanning microscopy wherein an image of a sample is generated by scanning and confocal imaging of a spot or spot group spot and means are provided for scanned point or dot group sample illumination.
  • Confocal laser scanning microscopes of the type mentioned above are known in the art, for example, reference is made to DE 197 02 753 A1 for this purpose.
  • microscopic structures in particular confocal laser scanning microscopes, have increasingly been used for spectroscopic recording techniques. In this way, it is possible to measure the spectroscopic properties of a selected sample area destructive and non-contact.
  • the confocal optical microscopy thereby allows the selective detection of optical signals which are generated within a diffraction-limited confocal volume whose size is in the micrometer range.
  • Laser scanning microscopes with scanning laser beams and / or sample feeding units can produce two- or three-dimensional representations of the examined sample with high spatial resolution. This feature has made confocal laser scanning microscopy the norm for fluorescent biomedical specimens.
  • DE 197 02 753 A1 also proposes to carry out a transmission measurement on the sample.
  • a detector can be activated, which lies with respect to the illumination direction of the scanned laser radiation under the sample and which receives the transmitted portion of the incident on the scanner point radiation.
  • a so-called "transmitted light scan” is realized.
  • the optical integration of the detector located below the sample poses certain difficulties, especially since the microscope part of the laser scanning microscope usually also provides an optical insight for an observer. This has the consequence that a switch between the Illumination is required for normal microscopy and the separate detector for the transmitted light scan.
  • the invention has the object of developing a microscope of the type mentioned so that transmission measurements on the sample can be made without increased effort.
  • This object is achieved according to the invention with a laser scanning microscope of the type mentioned, in which additionally a wide field illumination source is provided which illuminates the sample, and in which the control unit so controls the scanning arrangement during operation of the wide field illumination source and the spot detector array reads out so that an image the far-field illuminated sample (23) is obtained.
  • the invention is further achieved by a method of laser scanning microscopy of the type mentioned in the beginning, in which the sample is far field illuminated and is imaged by scanning the dot or dot group spot.
  • the invention thus now uses for the first time a wide-field illumination in combination with a scanned detection. With this surprisingly simple measure, a separate detector is dispensable. At the same time one achieves a wealth of advantages.
  • Radiation sources that are already present on the laser scanning microscope for normal optical observation can be used for the far-field illumination.
  • a switching mechanism is no longer needed. Overall, this results in a structural simplification.
  • the far-field illumination source will realize transmitted light illumination of the sample.
  • a wide-field incident illumination is possible, for example, to perform epi-fluorescence measurements or reflection measurements. You can also realize both modes (incident and transmitted light) simultaneously.
  • the depth discriminating ability of the confocal detector arrangement allows a depth-resolved transmission measurement for the first time.
  • the spot detector arrangement By scanning the far-field illuminated sample with the scanned detectors, a spectral analysis capability of the detector arrangement present in a laser scanning microscope can also be utilized in the transmission mode, which leads to a better sample characterization. Therefore, a development is preferred in which the spot detector arrangement has a plurality of spectral channels.
  • the wide field illumination can be operated independently of the scanned spot illumination.
  • the control unit may also initiate simultaneous operation in which the sample is then analyzed simultaneously in both transmission and conventional fluorescence modes.
  • control unit can read various spectral channels appropriately, so that in some spectral channels fluorescence information about the sample, obtained in other spectral channels transmission information. Appropriately combining this information, for example in a superimposed image, gives a conventional system superior sample analysis. It is therefore preferred that the control unit controls the spot illumination arrangement and the wide field illumination source simultaneously in operation and reads out the spectral channels of the spot detector arrangement appropriately.
  • Another advantage of the approach according to the invention is that a transmitted-light scan is now also possible at several points at the same time, which conventional, separate detectors arranged under the sample did not allow for lack of suitable spatial resolution.
  • the use of a multipoint or dot group scanner in transmitted light mode which is now opened by the invention, alleviates any problems caused by temporal fluctuations of the wide field illumination, since this can be compensated for by suitable lengthening of the integration time in multipoint or point group systems. It is therefore preferred that the far field illumination and the scanned spot or dot group illumination be made simultaneously.
  • dot group is meant any arrangement of several points, in particular in the form of a line which confocally illuminates and images the laser scanning microscope.
  • the spot detector arrangement realizes a confocal point group image, e.g. B. with at least one Nipkow disc and at least one matrix detector.
  • the spot detector arrangement may also use a confocal slit diaphragm with a line detector if a line dinet as a point group.
  • the use of wide field illumination finally opens up completely new contrasting methods for transmitted light measurement. All contrasting techniques are now possible, as known in the art for conventional optical light microscopes.
  • the far-field illumination source has a condenser into which contrasting means are switchable. For example, you can realize dark field lighting by a suitable annular aperture is arranged in the condenser.
  • FIG. 1 schematically shows a laser scanning microscope 1 which is composed essentially of five components: a radiation source module 2 which generates excitation radiation for laser scanning microscopy, a scanning module 3 which conditions the excitation radiation and deflects it appropriately for scanning via a sample, a microscope module 4, which directs the scanning radiation provided by the scanning module in a microscopic beam path to a sample, and a detector module 5, which receives and detects optical radiation from the sample.
  • the detector module 5 can, as shown in FIG. 1, be embodied spectrally with multiple channels.
  • the radiation source module 2 generates illumination radiation which is suitable for laser scanning microscopy, that is to say in particular radiation which can trigger fluorescence.
  • the radiation source module has a plurality of radiation sources for this purpose.
  • two lasers 6 and 7 are provided in the radiation source module 2, which are each followed by a light valve 8 and an attenuator 9 and couple their radiation via a coupling point 10 in an optical fiber 11.
  • the light valve 8 acts as a deflector with which a beam shutoff can be effected without having to switch off the operation of the laser in the laser unit 6 or 7 itself.
  • the light valve 8 is formed, for example, as AOTF, which deflects the laser beam before coupling into the optical fiber 11 in the direction of a light trap, not shown.
  • the laser unit 6 has three lasers B, C, D, whereas the laser unit 7 contains only one laser A.
  • the representation in FIGS. 6 and 7 is thus exemplary for a combination of single and multi-wavelength lasers which are coupled individually or also jointly to one or more fibers. The coupling can also take place simultaneously via a plurality of fibers, the radiation of which is later mixed after passing through an adjustment optics by means of color combiner. It is thus possible to use a wide variety of wavelengths or ranges for the excitation radiation.
  • the coupled into the optical fiber 11 radiation is merged by means of slidable collimating optics 12 and 13 via beam combination mirror 14, 15.
  • the collimators 12, 13 ensure that the radiation supplied by the radiation source module 2 to the scanning module 3 is collimated into an infinite beam path. This is in each case advantageously carried out with a single lens, which has a focusing function by displacement along the optical axis under the control (not shown) of a central drive unit in that the distance between collimator 12, 13 and the respective end of the optical fiber is variable.
  • This illumination beam serves as excitation radiation and is conducted via a main color splitter 17 to a scanner 18.
  • a main color splitter 17 On the main color divider will be discussed later, here's merely mentions that it has the function of separating sample radiation returning from the microscope module 4 from the excitation radiation.
  • the scanner deflects the beam biaxially, after which it is focused by a scanning objective 19 and a tube lens 20 and an objective 21 into a focus 22 which lies in a preparation or in a sample 23.
  • the optical imaging takes place so that the sample 23 is illuminated in a focal point with excitation radiation.
  • the main color splitter 17 passes the fluorescence radiation lying in wavelength ranges other than the excitation radiation, so that it can be deflected via a deflection mirror 24 in the detector module 5 and then analyzed.
  • the detector module 5 in the embodiment of Figure 1 has four spectral channels, i. the fluorescence radiation coming from the deflecting mirror 24 is divided into four spectral channels by means of secondary color splitters 25, 25a, 25b, 25c.
  • Each spectral channel has a confocal pinhole diaphragm 26 which realizes a confocal image with respect to the sample 23 and whose magnitude determines the depth of focus with which the radiation can be detected.
  • the geometry of the diaphragm 26 thus determines the cutting plane within the (thick) preparation from which radiation is detected.
  • the aperture 26 is still a block filter 27 downstream, which blocks unwanted, in the detector module 5 reached excitation radiation.
  • the radiation separated in such a way and originating from a certain depth section is then analyzed by a suitable detector 28.
  • Analogous to the described first color channel and the other spectral detection channels are constructed, which also include a Pinholeblende 26a and 26b, 26c, a block filter 27a and 27b, 27c, and a detector 28a and 28b, 28c.
  • the laser scanning microscope 1 of FIG. 1 also makes possible a further measuring mode of operation in the illustrated embodiment, which is optional in this respect.
  • a halogen lamp 29 is provided, whose radiation is directed via a lamp optics 30 and a condenser 31 counter to the viewing direction of the scanning optics 19 to the sample 23 in a wide-field illumination. Shares transmitted by this illumination become also scanned by the lens 21, the tube lens 20, the scanning lens 19 and the scanner 18 in the scanning process and spectrally analyzed by the main color divider 17 and the sub-color divider in the detector module 5.
  • the detection via the scanner 18 causes the spatial resolution in the form of sample scanning, and at the same time a wide-field illumination via the halogen lamp 29 is possible.
  • the same concept can also be used for evaluating retroreflected radiation and epifluorescence radiation in which illumination radiation is coupled into the tube of the microscope module 4 via a mercury vapor lamp 34 with lamp optics 35 at a beam splitter 36. This radiation then passes through the objective 21 to the sample 23.
  • the illumination takes place without the assistance of the scanner 18.
  • the detection takes place via the scanning optics 19 and the scanner 18 in the detector module 5.
  • the detector module 5 thus has a for this development Dual function, on the one hand it serves as a detector for scanned irradiated excitation radiation.
  • the detector module 5 acts as a spatially resolving detector when not further structured radiation is irradiated to the sample, namely either as far field illumination from below or via the lens 21.
  • the scanner 18 has double effect, since he spatial resolution by punctiform sampling of Not only in the case of punctiform irradiated excitation radiation, but also in far-field illumination.
  • the laser scanning microscope 1 of Figure 1 now allows a combination operation in which both point or point group irradiated excitation radiation from the radiation source module 2 and wide field illumination from the halogen lamp 29 and the mercury vapor lamp 34 is directed to the sample 23 and by means of the scanner 18 and the detector module 5 a corresponding point or point group-shaped scanning of such a multiple irradiated sample is achieved.
  • the auxiliary color splitters 25 to 25c the classical transmission or reflection microscopy can thus be combined with laser fluorescence measurement.
  • the image information thus obtained by scanning by means of the evaluation of the signals of the detectors 28 to 28c can then be evaluated or displayed independently or superimposed.
  • FIG. 1 The construction of FIG. 1 is to be understood as an example only with regard to the detector module 5.
  • Figure 2 showed an alternative construction, in which instead of a point-shaped focus 22, a line-shaped confocal image is used. These are in the Detector module 5, which is now equipped for example with two spectral channels, instead of the Pinholeblenden 26 slot apertures 26 'provided.
  • this slit-shaped partial confocal image also requires a corresponding illumination of the sample 23 by the laser radiation module 3 with a focal line. Therefore, in the construction of FIG. 2, the beam combining mirrors 14 and 15 are additionally followed by a beam-shaping unit 16 which, from the rotationally symmetrical, Gaussian-profiled laser beam delivered by the lasers, forms a line-shaped beam which is no longer rotationally symmetrical, but in cross-section to produce a rectangularly illuminated field is suitable, manufactures.
  • the scanner 18 thus deflects an illumination beam, which can be designated as a line, as excitation radiation over the sample 23. With sufficient length of the focal line, the scanner 18 can then, as Figure 2 also shows, be limited to a uniaxial deflection.
  • a confocal slot aperture in the detector module 5 is only exemplary. In principle, arbitrary multipoint arrangements, such as point cloud or Nipkow disk concepts, can be used. It is essential, however, that the detector 28 is then spatially resolving so that a parallel detection of several sample points takes place during the passage of the scanner.
  • the main color splitter 17 and the sub color splitter 25 are appropriately set. This also allows both types of illumination, i. Wide-field illumination from below and illumination by the objective 21, at the same time, if the color splitters are designed as suitable dichroic. Any combinations with scanned illumination from the radiation source module 2 are also possible. A corresponding superimposed graphical representation of the evaluated signals then offers superior image information over conventional concepts.
  • a confocal multipoint mapping e.g. B. a line scanner
  • a spectral multi-channel detection allows highly parallel data acquisition. It is one Image acquisition rate of over 200 images per second achievable and given a laser scanning microscopes previously unrealized real-time capability.
  • the laser scanning microscope 1 also enables a highly sensitive detection of particularly weak signal intensities. Compared with a confocal single-point laser scanning microscope with the same image acquisition time, with the same area imaged in the sample, the same field of view and the same laser power per pixel one by one
  • Factor ⁇ n improves the signal-to-noise ratio if n denotes the number of multipoint mappings. For a detector line, a value of 500 to 2,000 is typical.
  • the beam source module 2 of the laser scanning microscope 1 fulfills the necessary prerequisite, namely that the multi-point illumination, z. B. the lighting line, the z. B. is provided by the beam forming unit 16, which has n-fold performance, such as the laser focus of a comparable confocal single-point scanner.
  • sample loading i. the amount of radiation which is exposed to the sample and which can lead to the bleaching of the sample can be reduced by a factor of n, if the radiation power previously used in a confocal single-point scanner is now limited to several points, e.g. B. the line is distributed.
  • the multi-point scanning laser scanning microscope makes it possible compared to confocal single-point scanners, low-intensity signals of sensitive sample substances with the same signal / noise ratio and the same sample load by the factor n faster, with the same recording time with a factor ⁇ n improved signal / noise ratio or at the same recording time with the same signal-to-noise ratio and a smaller sample load by a factor of n.
  • FIG. 3 schematically shows an exemplary beam path for the laser scanning microscope 1 of FIG. 1 or 2 in construction with a line scanner.
  • the point group is realized here as a line. Accordingly, the illuminated spot is a line.
  • components already appearing there are designated by the same reference numerals, and reference is therefore made, at least in part, to FIGS. 1 and 2 with regard to the description.
  • FIG. 3 the illumination beam path B is illustrated by solid lines, whereas the detection beam path D is symbolized by dashed lines.
  • Figure 3 shows that the sample 23 is illuminated in two ways. On the one hand takes place via the halogen lamp 29, the lamp optics 30 and the condenser 31, for the in Figure 3, two optics 31a, 31b are drawn, a wide-field illumination. On the other hand, the laser 7 causes a line-shaped illumination, which is why the illustration in FIG. 3 has no focus in the sample in the illustrated cutting direction.
  • the scanner 18 deflects a line across the sample 23 and forms it confocally on a confocal aperture 26 associated with the spot image, which is designed as a slit diaphragm due to the linear spot.
  • the coherence of the linearly incident excitation radiation is canceled by the fluorescence excitation, since the fluorescence is an incoherent interaction process.
  • the dimension of the dye molecules is below the optical resolution of the microscope. Each dye molecule radiates as a spotlight independently of the other sample locations and fills the entire lens pupil. This results in the dashed lines drawn detection beam path in Figure 3.
  • the thus realizable as a focal line spot-shaped illumination is separated from the detection beam D via the main color divider 17.
  • This main color divider is designed as a streak mirror according to DE 10257237 A1 in the present embodiment. It is shown in plan view in FIG.
  • Such a strip mirror which has a highly reflective part HR and a high-transmission part HT, acts as a spectrally independent main divider. It lies, as can be seen, in a pupil plane of the scan arrangement in which reflected in the sample plane, d. H. coherent illumination radiation line-shaped (punctiform in a single-point detector) is focused. In contrast, incoherent signal radiation to be detected fills the entire pupil plane and is essentially transmitted through the highly reflective part HT.
  • a "color splitter” is therefore also understood to mean a non-spectrally acting splitter.
  • a field stop is provided between the lamp optics 30 and the condenser 31a, 31b in order to be able to set the illuminated area.
  • an aperture diaphragm A1 can be switched into the condenser 31a, 31b. It lies in a conjugate position to the pupil planes of the laser scanning microscope. These pupil planes are the pupil plane P1, the plane in which the scanner 18 lies, and the plane in which the main color splitter 17 is arranged.
  • various optical elements can now be used to use contrasting methods known from classical microscopy, such as dark field, phase contrast, VAREL contrast or differential interference contrast.
  • Suitable apertures A1 or elements to be introduced into the pupil plane P1 are explained, for example, in the publication "Microscopy from the very beginning", Carl Zeiss Microscopy, D-07740 Jena, 1997, pages 18-23. The content of this company publication is explicitly included here. Of course, not only the pupil plane P1 is suitable for such contrasting interventions. Other pupil levels are also suitable.
  • the intervention could also take place in the vicinity of the main color splitter 17 or by means of relay optics after the auxiliary color splitter 25 in one (or more) spectral channels of the detector beam path.
  • the beam path of FIG. 3 also has a relay optics RO which, in addition to the intermediate image plane ZB1 between scanning optics 19 and tube lens 20, provides a further intermediate image plane ZB2 for the illumination beam path.
  • a third intermediate image plane ZB3 of the illumination beam path is located in front of the main divider 17, so that a pupil plane for color splitters is created. If you want to keep the structure compact, the relay optics RO can also be omitted.
  • the invention described represents a significant expansion of the application possibilities of fast confocal laser scanning microscopes. The significance of such a further development can be read off from the standard cell biological literature and the fast cellular and subcellular processes 1 described therein and the examination methods used with a plurality of dyes 2 .
  • the invention has particular importance for the following processes and processes:

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
EP04023492A 2004-07-16 2004-10-01 Microscope optique à balayage avec une répartition ponctuelle de la lumière et son utilisation Withdrawn EP1617249A1 (fr)

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DE102004034994A DE102004034994A1 (de) 2004-07-16 2004-07-16 Lichtrastermikroskop mit punktförmiger Lichtquellenverteilung und Verwendung

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114273775A (zh) * 2021-12-23 2022-04-05 中国科学院西安光学精密机械研究所 一种基于振镜系统的激光光束抖动校正装置与方法

Families Citing this family (2)

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Publication number Priority date Publication date Assignee Title
DE102004034976A1 (de) * 2004-07-16 2006-02-16 Carl Zeiss Jena Gmbh Lichtrastermikroskop und Verwendung
DE102011013614A1 (de) * 2011-03-08 2012-09-13 Carl Zeiss Microimaging Gmbh Laser-Scanning-Mikroskop und Verfahren zu seinem Betrieb

Citations (3)

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WO1988007695A1 (fr) * 1987-03-27 1988-10-06 The Board Of Trustees Of The Leland Stanford Junio Microscope optique confocal a balayage
US6388807B1 (en) * 1999-10-12 2002-05-14 Leica Microsystems Heidelberg Gmbh Confocal laser scanning microscope
DE10257237A1 (de) * 2001-12-10 2003-06-18 Zeiss Carl Jena Gmbh Anordnung zur optischen Erfassung von in einer Probe angeregter und/oder rückgestreuter Lichtstrahlung

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US5932871A (en) * 1995-11-08 1999-08-03 Olympus Optical Co., Ltd. Microscope having a confocal point and a non-confocal point, and a confocal point detect method applied thereto
US6167173A (en) * 1997-01-27 2000-12-26 Carl Zeiss Jena Gmbh Laser scanning microscope
JP3816632B2 (ja) * 1997-05-14 2006-08-30 オリンパス株式会社 走査型顕微鏡
US6248988B1 (en) * 1998-05-05 2001-06-19 Kla-Tencor Corporation Conventional and confocal multi-spot scanning optical microscope

Patent Citations (3)

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WO1988007695A1 (fr) * 1987-03-27 1988-10-06 The Board Of Trustees Of The Leland Stanford Junio Microscope optique confocal a balayage
US6388807B1 (en) * 1999-10-12 2002-05-14 Leica Microsystems Heidelberg Gmbh Confocal laser scanning microscope
DE10257237A1 (de) * 2001-12-10 2003-06-18 Zeiss Carl Jena Gmbh Anordnung zur optischen Erfassung von in einer Probe angeregter und/oder rückgestreuter Lichtstrahlung

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114273775A (zh) * 2021-12-23 2022-04-05 中国科学院西安光学精密机械研究所 一种基于振镜系统的激光光束抖动校正装置与方法

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US20060012856A1 (en) 2006-01-19
DE102004034994A1 (de) 2006-02-02
GB0508771D0 (en) 2005-06-08

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